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Titanium is widely used in implant materials, while excessive fluoride may have negative effects on the osseointegration between the titanium and osteoblasts. Although the underlying mechanisms are still not clear, the mitogen-activated protein kinase (MAPK) or Yes-associated protein (YAP) signaling pathways are thought to be involved. This study evaluated the role of Hippo/YAP and MAPK signaling pathway in osteoblast behaviors under excessive fluoride exposure in vitro and in vivo. Commercially pure Ti (cp-Ti) samples were exposed to fluoride (0, 0.1, and 1.0 mM NaF) for 7 days. Cell adhesion was observed using a laser scanning confocal microscope. Cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively. The expressions of osteoblast markers and key molecules in MAPK and YAP pathway were detected by Western blot. In vivo studies were evaluated by histology methods in C57/BL6 mice model. Our results showed that 1.0 mM NaF destroyed the passivation film on cp-Ti surface, which further inhibited the osteoblast adhesion and spreading. Meanwhile, compared to other groups, 1.0 mM NaF led to a remarkable reduction in cell viability (P < 0.05), as well as increased apoptosis (P < 0.05) and downregulation of osteogenesis protein expression (P < 0.05). MAPK and YAP signaling pathways were also activated under 1.0 mM NaF exposure, and JNK seemed to regulate YAP phosphorylation in response to NaF impacts on osteoblasts. In vivo fluorosis mouse model further indicated that 100 ppm NaF group (high fluoride group) increased bone resorption and inhibited the nuclear translocation of YAP. The osteoblast behaviors were negatively altered under excessive fluoride, and MAPK/JNK axis contributed to YAP signaling activation in regulating NaF-induced osteoblast behaviors. KEY MESSAGES: • Excessive fluoride inhibited osteoblast behaviors and bone formation. • YAP and MAPK signaling pathways were activated in osteoblasts under fluoride exposure. • Fluoride regulated osteoblast behaviors via the cross-talk between YAP and MAPK.

The aim of this study was to explore the changes in the characteristics of titanium surface and the osteoblast-titanium interactions under cigarette smoke extract (CSE) exposure. In this study, CSE was used to simulate the oral liquid environment around the implant under cigarette smoke exposure. Titanium samples were immersed in CSE to explore the changes in the characteristics of titanium surface. The physical properties of titanium surface were measured, including surface micromorphology, surface elemental composition, roughness, and surface hydrophilicity. MC3T3-E1 cells were cultured on the titanium surface in vitro under different concentrations of CSE exposure, and cell adhesion, cell proliferation, and osteogenic differentiation were observed. The surface micromorphology and elemental composition of titanium surface changed under CSE exposure. No obvious changes were found in the surface roughness and the hydrophilicity of titanium samples. Moreover, the results of in vitro study showed that CSE exposure downregulated the cell spreading, proliferation, and osteogenic differentiation of MC3T3-E1 cells on the titanium surface. It could be speculated that some carbon-containing compounds from CSE adsorbed on the titanium surface and the osteoblast-titanium interactions were influenced under CSE exposure. It is hoped that these results could provide valuable information for further studies on smoking-mediated inhibition of implants osseointegration.

Titanium (Ti) and its corresponding alloys have been widely applied in dental and orthopedic implants. Owing to abrasion and corrosion of implants in the unfavorable electrolytic aqueous environment of the host body, Ti ions could be released from implants and accumulated in local tissues. Recent studies have found that excessive Ti ions were toxic to osteoblasts in adjacent bone tissues and subsequently influenced long-term effects on implant prostheses. However, the potential molecular mechanisms underlying the damage to osteoblasts induced by Ti ions remained unclear. Hippo signaling has been confirmed to be involved in organ size and tissue regeneration in many organs, while its roles in osteoblasts differentiation and bone repair remained elusive. Therefore, we hypothesize that YAP, a regulator of Hippo pathway, inhibited osteoblast growth, skeletal development and bone repair, as well as excessive Ti ions promoted the progression of YAP activation. This study aimed to explore the role of Hippo/YAP signaling pathway in the biotoxicity effect of Ti ions on osteoblast behaviors. Here, we confirmed that 10 ppm Ti ions, a minimum concentration gradient previously reported that was capable of suppressing osteoblasts growth, induced nuclear expression of YAP in osteoblasts in our study. Furthermore, 10 ppm Ti ion-induced YAP activation was found to downregulate osteogenic differentiation of MC3T3-E1 cells. Most importantly, the hypothesis we proposed that knockdown of YAP did reverse the inhibitory effect of 10 ppm Ti ions on osteogenesis has been verified. Taken together, our work provides insights into the mechanism of which YAP is involved in regulating osteoblast behaviors under the effect of Ti ions, which may help to develop therapeutic applications for Ti implant failures and peri-implantitis., (Copyright © 2018 John Wiley & Sons, Ltd.)